Photoaffinity labeling has been an important method for the direct elucidation of intermolecular interactions in biological systems (Bayley, H., Photogenerated Reagents in Biochemistry and Molecular Biology, Elsevier, Amsterdam (1983)) since its introduction by Westheimer and coworkers (Singh, A., Thornton, E. R. and Westheimer, F. H., J. Biol. Chem. 237, 3006-3008 (1962)) more than three decades ago. A variety of photophores have been employed, most relying on photoconversion of diazo compounds, azides, or diazirines to nitrenes or carbenes (Bayley, H., Photogenerated Reagents in Biochemistry and Molecular Biology (1983), Elsevier, Amsterdam. These reactive intermediates have the disadvantage of reacting rapidly with water, leading to very low photoinsertion yields in most cases. The introduction of benzophenones as photoactivatable reagents by Galardy, R. E., Craig, L. C. and Printz, N. P., Nature New Biol. 242, 127-128 (1973) led to the recognition of a significant advantage of this photophore over the nitrene and carbene precursors: the excited state of the benzophenones, a triplet biradical, is essentially inert toward water. Other advantages of this photophore over previous ones include stability to ambient light, activation at longer wavelengths (thus minimizing photolytic damage to proteins), and a much wider range of chemical stability. The photochemistry of benzophenones has been reviewed (Dorman, G., and Prestwich, G. D., Biochemistry 33, 5661-5673 (1994)). Introduction of the amino acid 4-benzoylphenylalanine (BPA.sup.1) by Kauer, J. C., Erickson-Vitanen, S. Wolfe, H. R. J. and DeGrado, W. F., J. Biol. Chem. 261:10695-10400 (1986) allowed direct incorporation of a benzophenone photophore at a defined position into peptides by solid-phase synthesis, bringing a significant advance in the application of photoaffinity labeling to the study of peptide-protein interactions (Adams, A. E., Pines, M., Nakamoto, C., Behar, V., Yang, Q. M., Bessalle, R., Chorev, M., Rosenblatt, M., Levine, M. A. and Suva, L. J., Biochemistry 34, 10553-105539 (1995); Behar, V., Pines, M., Nakamoto, C., Greenberg, Z., Bisello, A., Stueckle, S. M., Bessalle, R., Usdin, T. B., Chorev, M., Rosenblatt, M. and Suva., L. J., Endocrinology 137, 2748-2757 (1996); Blanton, M. P., Li, Y. M., Stimson, E. R., Maggio, J. E. and Cohen, J. B., Mol. Pharmacol. 46, 1048-1055 (1994); Bosse, R., Servant, G., Zhou, L.-M., Guillemette, G. and Escher, E., Regul. Peptides 44, 215-223 (1993); Boyd, N. D., Macdonald, S. G., Kage, R., Luber-Narod, J. and Leeman, S. E., Ann. New York Acad. Sci. 632, 79-93 (1991a); Boyd, N. D., White C. F., Cerpa, R, Kaiser, E. T. and Leeman, S. E., Biochemistry 30, 336-342 (1991b); Boyd, N. D., Kage, R. K. and Leeman, S. E., The Tachykinin Receptors (Buck, S. H., ed) pp. 219-236, Humana Press, Totowa, N.J. (1994); Boyd, N. D., Kage, R. K., Dumas, J. J., Krause, J. E. and Leeman, S. E., Proc. Natl. Acad. Sci. USA 93, 433-437 (1996); Gao, Z.-H, Zhi, G., Herring, B. P., Moomaw, Deogny, L., Slaughter, C. A. and Stull, J. T., J. Biol. Chem. 270, 10125-10135 (1995); Garcia, P., Shoelson, S. E., Drew, J. S. and Miller, W. T., J. Biol. Chem. 269, 30574-30579 (1994); Gergel, J. R., McNamara, D. J., Dobrusin, E. M., Zhu, G., Saltiel, A. R., and Miller, W. T., Biochemistry 33, 14671-14678 (1994); Kage, R. K., Leeman, S. E. and Boyd, N. D., J. Neurochem. 60, 347-351 (1993); Kauer, J. C., Erickson-Vitanen, S. Wolfe, H. R. J. and DeGrado, W. F., J. Biol. Chem. 261, 10695-10700 (1986); Li, Y.-M., Marnerakis, M., Stimson, E. R. and Maggio, J. E., J. Biol. Chem. 270, 1213-1220 (1995a); Macdonald, S. G., Dumas, J. J. and Boyd, N. D., Biochemistry 35, 2909-2916 (1996); McNicoll, N., Escher, E. Wilkes, B. C., Schiller, P. W., Ong, H. and DeLean, A., Biochemistry 31, 4487-4493 (1992); Miller, W. T. and Kaiser, E. T., Proc. Natl. Acad. Sci. USA 85, 5429-5433 (1988); O'Neil, K. T., Erickson-Vitanen, S. and DeGrado, W. F., J. Biol. Chem. 264, 14571-14578 (1989); Servant, G., Boulay, G., Bosse, R., Escher, E. and Guillemette, G., Mol. Pharmacol. 43, 677-682 (1993); Williams, K. P. and Shoelson, S. E., J. Biol. Chem. 268, 5361-5364 (1993); Zhang, Z.-Y., Walsh, A. B., Wu, L., McNamara, D. J., Dobrusin, E. M. and Miller, W. T., J. Biol. Chem. 271, 5386-5392 (1996)).
The large majority of receptors for bioactive peptides transduce signals through guanine nucleotide binding proteins. The question of which region(s) of a G-protein linked receptor interact with which region(s) of its peptide agonist has been difficult to approach. With neuropeptide ligands such as substance P (MW.apprxeq.3 kDa), many contacts between ligand and receptor are involved as compared to much smaller non-peptide ligands. Thus, useful data from mutagenesis experiments are more difficult to acquire because of the extremely large number of mutants required for thorough analysis, and because mutations causing loss of function could reflect a change in receptor conformation distant from the binding site (Huang, R. R., Vicario, P. P., Strader, C. D. and Fong, T. M., Biochemistry 34, 10048-10055 (1995)). In contrast, photoaffinity labeling offers a uniquely powerful approach by directly identifying regions of the receptor in close contact with an identified amino acid of the agonist.
Photolabeling of substance P receptor (SPR, also known as NK-1R, a member of the G-protein coupled receptor family involved in pain modulation and inflammation (Pernow, B., Pharmacol. Rev. 35, 85-141 (1983); Otsuka, M. and Yoshioka, K., Physiol. Rev. 73, 229-308 (1993)) by a substance P (SP) analog is desirable for several reasons. SP, a member of the tachykinin peptide family, is a high affinity ligand (0.5-1 nM) with a single binding site on SPR. SPR has been cloned from several species (human, mouse, rat, and guinea pig) and shows a high degree of sequence homology between them (Gerard, N. P., Bao, L., He, X. P. and Gerard, C., Regul. Peptides 43, 21-35 (1993)), but expression levels remain low and purification is difficult. Mutagenesis studies of SPR have suggested that both extracellular and transmembrane domains are important for agonist binding (e.g., Cascieri, M. A., Macleod, A. M., Underwood, D., Shiao, L. L., Ber, E., Sadowski, S., Yu, H., Merchant, K. J., Swain, C. J., Strader, C. D and Fong, T. M., J. Biol. Chem. 269, 6587-6591 (1994); Fong, T. M., Huang, R. R. C. and Strader, C. D., J. Biol. Chem. 267, 25664-25672 (1992a); Fong, T. M., Yu, H., Huang, R. R. C. and Strader, C. D., Biochemistry 31, 11806-11811 (1992b); Fong, T. M., Cascieri, M. A., Yu, H., Bansal, A., Swain, C. and Strader, C. D., Nature 362, 350-353 (1993); Fong, T. M., Yu, H., Cascieri, M. A., Underwood, D., Swain, C. J. and Strader, C. D., J. Biol. Chem. 269, 2728-2732 (1994a); Fong, T. M., Yu, H., Cascieri, M. A., Underwood, D., Swain, C. J. and Strader, C. D., J. Biol. Chem. 269, 14957-14961 (1994b); Gether, U., Johansen, T. E. and Schwartz, T. W., J. Biol. Chem. 268, 7893-7898 (1993a); Gether, U., Johansen, T. E., Snider, R. M., Lowe, J. A., III, Nakanishi, S. and Schwartz, T. W., Nature 368, 345-347 (1993b); Gether, U., Yokota, Y., Edmonds-Alt., X., Breliere, J. C., Lowe, J. A., III, Snider, R. M., Nakanishi, S., and Schwartz, T. W., Proc. Natl. Acad. Sci. USA 90, 6194-6198 (1993c); Gether, U., Edmonds-Alt., X., Breliere, J. C., Fuji, T., Hagiwara, D., Pradier, L., Garret, C., Johansen, T. E., and Schwartz, T. W., Mol. Pharmacol. 45, 500-508 (1994); Huang, R. R. C., Yu, H., Strader, C. D. and Fong, T. M., Mol. Pharmacol. 45, 690-695 (1994a); Huang, R. R. C., Yu, H., Strader, C. D. and Fong, T. M., Biochemistry 33, 3007-3013 (1994b); Jensen, C. J., Gerard, N. P., Schwartz, T. W. and Gether, U., Mol. Pharmacol. 45, 294-299 (1994); Sachais, B. S., Snider, R. M., Lowe, J. A., III and Krause, J. E., J. Biol. Chem. 268, 2319-2323 (1993); Yokota, Y., Akazawa, C., Ohkubo, H. and Nakanishi, S., EMBO J. 11, 3585-3591 (1992); Zoffman, S., Gether, U. and Schwartz, T. W. , FEBS Lett. 336, 506-510 (1993).
Benzoylphenylalanine sparked a revolution in photoaffinity labeling (Dorman and Prestwich, 1994), but has the distinct disadvantage that the high specific activity radiolabel must be located distal to the photoactive residue in the primary structure. Furthermore, neither the BPA amino acid nor its PTH (phenylthiohydantoin) analog is detectable using standard amino acid analysis or Edman sequencing protocols (Kauer et al., 1986), making identification of the photoinsertion site difficult. Thus, there is a need for new photolabeling reagents which overcome these shortcomings.